ABSTRACT Infectious diseases continue to pose a major threat to worldwide human health and lead to significant morbidity, mortality and healthcare costs. Viral respiratory tract infection (VRTI) is the most common illness in humans, and non-influenza-related VTRI costs amount to over $40 billion annually in the United States alone. Respiratory syncytial virus (RSV) is among the leading causes of pediatric death secondary to pneumonia worldwide. Rapid diagnosis is critical for early and accurate treatment, as well as initiation and reduction of transmission, eventually leading to reduced hospital stays, antibiotic over-prescription, and taxing limited laboratory resources. Current diagnostic methods rely on time-consuming laboratory-based tests including virus culture and polymerase chain reaction (PCR), and rapid diagnostic tests are not sufficiently sensitive as standalone diagnosis. This paradox raises the need for rapid and ultrasensitive diagnostic tests. The plasmonic coupling assay is a rapid colorimetric diagnostic test that changes color from red to purple when plasmonic gold nanoparticles (AuNPs) get in close proximity of each other following antibody binding or DNA hybridization. Despite its easy operation, the sensitivity of the colorimetric plasmonic coupling assay is limited. In this proposed work, we aim to dramatically improve the analytical sensitivity of the plasmonic coupling assay using an innovative digital nanobubble detection method. Specifically, we propose to directly detect RSV particles with antibody-conjugated AuNPs that recognize the RSV surface fusion protein. AuNPs bind to multiple RSV surface fusion proteins and lead to plasmonic coupling. Ultrashort laser pulse selectively activates coupled AuNPs due to their enhanced absorption compared with a single AuNP. This greater optical absorption leads to nanoscale cavitation bubbles, i.e. nanobubbles, which can be measured easily from their intense scattering. Single nanobubble generation leads to a sensitive digital detection with ?on? and ?off? signals. Our preliminary results suggests at least 3 orders of magnitude improvement in analytical sensitivity with a similar bioassay for nucleic acid detection. Our specific aims are: (1) to optimize the AuNP size and concentration for RSV detection using the digital nanobubble detection and establish the specificity and storage stability of the assay; and (2) to build and test a prototype device for automated digital nanobubble detection using micro lasers, compact optical components, and a capillary-driven flow device. The direct detection of virus particles eliminates the need for extensive sample preparation such as nucleic acid extraction. This is a high-reward project with a bold idea and corresponding robust preliminary data to address a major healthcare need for rapid and ultrasensitive diagnostic platforms for RSV and possibly other VTRI. The ultimate goal of our project is to develop rapid and ultrasensitive diagnostic tests with potential to replace current viral culture and PCR based laboratory tests.